The present invention relates to compositions and crystals of a hepatitis C virus RNA dependent RNA polymerase called NS5B and to methods of producing such crystals. This invention relates to methods of using the structure coordinates of hepatitis C virus NS5B to solve the structure of homologous NS5B proteins or complexes containing the NS5B protein.
Infection by hepatitis C virus (HCV) is a compelling human medical problem. HCV is recognized as the causative agent for most cases of non-A and non-B hepatitis, with an estimated human seroprevalence of 1% globally [Choo, et al., Science, 244:359-362 (1989); Kuo, et al., Science, 244:362-364 (1989); Purcell, FEMS Microbiology Reviews; 14:181-191 (1994); Van der Poel. Current Studies in Hematology and Blood Transfusion, H. W. Reesink, Ed., (Basel: Karger), pp. 137-163 (1994)]. Four million individuals may be infected in the United States alone [Alter, and Mast, Gastroenterol. Clin. North Am., 23:437-455 (1994)].
Upon first exposure to HCV, only about 20% of infected individuals develop acute hepatitis and appear to resolve the infection spontaneously. In the most instances (xcx9c80%), however, the virus establishes a chronic infection that persists for decades [Iwarson, FEMS Microbiology Reviews, 14: 201-204 (1994)]. This usually results in recurrent and progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma [Kew, FEMS Microbiology Reviews, 14: 211-219 (1994); Saito, et al., Proc. Natl. Acad. Sci. USA 87: 6547-6549 (1990)].
The HCV genome encodes a polyprotein of approximately 3000 amino acids [Choo, et al. Proc. Natl. Acad. Sci. USA, 88: 2451-2455 (1991); Kato, et al., Proc. Natl. Acad. Sci. USA, 87: 9524-9528 (1990); Takamizawa, et al., J. Virol., 65:1105-113 (1991)]. The HCV non-structural (NS) proteins provide catalytic machinery for viral replication. The NS proteins are derived by proteolytic cleavage of the polyprotein [Bartenschlager, et al., J. Virol., 67: 3835-3844 (1993); Grakoui, et al., J. Virol, 67: 2832-2843 (1993); Grakoui, et al., J. Virol., 67:1385-1395 (1993); Tomei, et al., J. Virol., 67:4017-4026 (1993)].
Until recently, the only therapy available for treating chronic HCV infection was interferon-xcex1 (IFN-xcex1). However, not all patients are responsive to IFN-xcex1 treatment. While combination therapy of IFN-xcex1 and ribavirin has significantly improved the clinical outcome, a need exists for more effective methods of treatment.
The NS5B RNA dependent RNA polymerase is considered a valuable target for antiviral agents. However, drug discovery efforts directed towards the NS5B protein have been hampered by the lack of structural information about NS5B. Such structural information would provide valuable information for discovery of HCV NS5B polymerase inhibitors. However, efforts to determine the structure of HCV NS5B polymerase have been hampered by difficulties in obtaining sufficient quantities of pure active enzyme and by poor solubility of the enzyme. There have been no crystals reported of any NS5B polymerase polypeptide. Thus, x-ray crystallographic analysis of such proteins has not previously been available.
The present invention addresses this need by providing, for the first time, compositions comprising a crystallized hepatitis C virus (HCV) NS5B polypeptide. Methods for obtaining purified and crystallized NS5B polypeptide are also provided. Such methods comprise solubilizing a NS5B polypeptide in a solution containing a protein stabilizing agent, subjecting the NS5B preparation to cation exchange chromatography, and allowing crystals to form in a precipitant solution containing a protein stabilizing agent and polyethylene glycol under conditions in which crystallization occurs.
The invention also provides a machine-readable data storage medium encoded with the structural coordinates of a NS5B polypeptide or a homologue thereof. Such a homologue contains backbone atoms having a root mean square deviation of equivalent Cxcex1 atoms of less than 3.0 xc3x85 when compared to the NS5B polypeptide.
The invention also provides a method for determining at least a portion of the three-dimensional structure of molecules or molecular complexes which contain at least some structurally similar features to a HCV NS5B polypeptide.
Still another aspect of the present invention comprises a method of selecting or optimizing a potential ligand or inhibitor by performing drug design with a three-dimensional structure determined for the crystal, preferably in conjunction with computer modeling. The potential ligand or inhibitor is then contacted with the NS5B polypeptide and the binding thereof is detected. If the ligand is a potential inhibitor of NS5B activity, the candidate drug may then be contacted with NS5B and the inhibition of its activity can be measured.
In another embodiment of the invention, a method of obtaining structural information concerning a molecular complex of unknown structure by using the structure coordinates set forth in Table 1 is provided. Such a method comprises the steps of: generating x-ray diffraction data from a crystal of said complex, and applying crystallographic phases derived from at least a portion of the structure coordinates set forth in Table 1 to said x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the unknown structure.